Prognostic Impact of AGR3 Protein Expression in Breast Cancer: A Systematic Review and Meta-analysis

Objective  To investigate the clinicopathological significance and prognosis of the expression of the anterior gradient 3 (AGR3) protein in women with breast cancer. Data Sources  The PubMed, CINAHL, EMBASE, Scopus, and Web of Science databases were searched for studies published in English and without restrictions regarding the year of publication. The search terms were: breast cancer AND anterior gradient 3 OR AGR3 expression . Study Selection  We included observational or interventional studies, studies on AGR3 protein expression by immunohistochemistry, and studies on invasive breast cancer. Case reports, studies with animals, and reviews were excluded. In total, 4 studies were included, containing 713 cases of breast cancer. Data Collection  Data were extracted on clinicopathological characteristics and survival. A meta-analysis of the prevalence of AGR3 expression was performed according to the clinicopathological characteristics, hazard ratios (HRs), and overall survival and disease-free survival. Data Synthesis  The expression of AGR3 was found in 62% of the cases, and it was associated with histological grade II, positivity of estrogen and progesterone receptors, low expression of ki67, recurrence or distant metastasis, and lumen subtypes. In patients with low and intermediate histological grades, AGR3 expression was associated with worse overall survival (HR: 2.39; 95% confidence interval [95%CI]: 0.628–4.159; p  = 0.008) and worse disease-free survival (HR: 3.856; 95%CI: 1.026–6.686; p  = 0.008). Conclusion  The AGR3 protein may be a biomarker for the early detection of breast cancer and predict prognosis in luminal subtypes. In addition, in patients with low and intermediate histological grades, AGR3 protein expression may indicate an unfavorable prognosis in relation to survival.


Introduction
Breast cancer is of great epidemiological relevance due to the high rates of mortality and morbidity in the world.In 2020, breast cancer represented the main cause of death due to cancer among women, affecting $ 2.3 million new cases. 1ith advances in large-scale techniques, gene expression signatures capable of stratifying breast cancer into molecular subtypes that aid in diagnosis, response to treatment, and prognosis have been proposed.Through these analyses, breast cancers have been stratified into four subtypes: luminal A, luminal B, human epidermal growth factor receptor-2 positive (HER2 þ ), and basal-like. 2,3Despite the translational application of the molecular stratification of breast cancer, many patients develop resistance to treatments and recurrence, which instigates research that seeks new biomarkers of prognosis and response to chemotherapy. 4nterior gradient 3 (AGR3) is a member of the protein disulfide isomerase (PDI) gene family.In recent years, the protein encoded by this gene has attracted the attention of researchers due to its role in the process of carcinogenesis. 51][12][13] Scientific evidence has suggested that AGR3 has prognostic value in ovarian and breast cancers. 6,14In ovarian cancer, AGR3 is upregulated in the serous 6 and clear-cell subtypes, 7 and high levels of AGR3 are a predictor of better survival. 6In breast cancer, AGR3 is considered a potential biomarker for early detection in blood and tissue 5,11 and for prognosis. 13he role of AGR3 in the clinic of breast cancer remains nuclear, 5,12 due to the limited studies that present the clinicopathological and prognostic relevance of this protein. 5owever, the evidence suggest that AGR3 may be associated with oncogenesis, and it has been pointed out as a potential therapeutic target and prognostic biomarker for patients with breast cancer.According to these precedents, the objective of the present systematic review and meta-analysis was to investigate the clinicopathological significance and intermediate histological grades, AGR3 protein expression may indicate an unfavorable prognosis in relation to survival.

Palavras-chave
prognosis of the expression of the AGR3 protein in women with breast cancer.

Materials and Methods
The present systematic review and meta-analysis was performed following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) 15 statement and the Meta-analysis of Observational Studies in Epidemiology (MOOSE) group. 16In adittion, the study was registered in the International Prospective Register of Systematic Reviews (PROSPERO) database (CRD42021244277).
An electronic search was performed on the PubMed, CINAHL, EMBASE, Scopus, and Web of Science databases.Searches on Google Scholar and the primary study reference list were also conducted to identify additional studies.The search terms were: breast cancer AND anterior gradient 3 OR AGR3 expression.The search strategies for each database are presented in chart 1.
The inclusion criteria were: 1) observational or interventional studies involving the expression of the AGR3 protein in women with breast cancer; 2) studies evaluating the prognostic capacity of the AGR3 protein expression by immunohistochemistry; and 3) studies on invasive breast cancer; moreover, there were no restrictions regarding language or the year of publication of the studies.The following were excluded: dissertations, theses, case reports, studies with animals, reviews, editorials, letters to the editor, and duplicate studies found in more than one database.
Titles and abstracts were read using the Rayyan (Rayyan Systems Inc., Cambridge, MA, United States) software.The studies retrieved were analyzed by the authors, the selected articles were read in full, and the inclusion and exclusion criteria were applied.Doubts and/or disagreements about the articles were discussed by the research team.
The following data were extracted: author and year of publication, study design, country, number of patients, age of the patients, methods of evaluation and results of AGR3 expression in women with breast cancer, and clinical results (clinicopathological characteristics and survival).The clinicopathological characteristics included: age, histological grade, estrogen receptor (ER), progesterone receptor (PR), HER2, K i À67, recurrence or distant metastasis, and molecular subtypes (luminal A, luminal B, HER2 þ , and triple-negative).The data collected on survival were: overall survival (OS) and disease-free survival (DFS).
The risk of bias was assessed using the Cochrane's Risk of Bias in Non-Randomized Studiesof Interventions (ROBINS-I) tool. 17Eight methodological domains were evaluated: 1) bias due to confounding; 2) bias in the selection of participants into the study; 3) bias in the measurement of interventions; 4) bias due to departures from the intended interventions; 5) bias due to missing data; 6) bias in the measurement of outcomes; 7) bias in the selection of the reported result; and 8) overall bias.Each domain was classified as presenting "low risk of bias," "moderate risk of bias," "serious risk of bias," and "critical risk of bias." The methodological quality of the studies was evaluated using the software application of the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) approach (https://gdt.gradepro.org/app/#), 18,19 20Thus, the quality of the evidence was classified into these aforementioned categories.Meta-analyses were conducted using the random effects model on coded data stratified by the expression of AGR3.The meta-analysis of prevalence of AGR3 expression was performed according to clinicopathological characteristics, hazard ratios (HRs) and OS and DFS analyses.The data were expressed graphically in forest plots, with estimates on the prevalence and HRs with 95% confidence intervals (95%CIs).The degree of heterogeneity among the studies was estimated by the statistical values of I 2 : < 25%low heterogeneity;, 25% to 50%moderate heterogeneity; and > 50% -high heterogeneity. 21Publication bias was assessed using the Egger test and funnel plot asymmetry.All analyses were performed using the STATA (StataCorp LLC, College Station, TX, United States) software, version 16.0.

Results
A total of 116 articles were identified in the 5 databases evaluated.After the careful process of screening and removing duplicates, 69 articles were selected and had their titles and abstracts read; then, 9 articles were selected for full-text reading, and 4 articles presented the eligibility criteria and were included in the present systematic review and metaanalysis. 5,10,11,13The article selection process is illustrated in a flowchart prepared in accordance with the PRISMA statement (►Fig.1).
The excluded articles and the reasons for the exclusions are presented in chart 2.
Four studies were evaluated using the GRADE approach and ROBINS-I.The GRADE score indicated that three studies showed moderate quality of evidence 5,11,13 and one study, showed poor quality (►Table 1). 10 The results of the risk of bias assessment are shown in ►Fig. 2. Prognostic Impact of AGR3 Protein Expression in Breast Cancer Moraes et al.
A funnel plot was developed to assess the publication bias (►Fig. 3).This analysis revealed a symmetrical pattern, and there was no evidence of a notable publication bias that could confuse the results.The Egger test ruled out the apparent bias in studies that analyzed the expression of AGR3 in women with breast cancer (p ¼ 0.105).
The present systematic review included four studies, 5,10,11,13 comprising a total of 713 cases of breast cancer from Germany, 11 the United Kingdom, 10 the Czech Republic, 5 and China. 13The characteristics of each study are shown in table 1.
The studies included showed results of the prevalence of AGR3 expression in women with breast cancer that were included in the meta-analysis. 5,10,11,13The prevalence of AGR3 expression was of 62%, as shown in ►Fig. 4.
Information about the association regarding AGR3 expression and OS 5,11,13 and DFS 5,13 in women with breast cancer are shown in ►Table 3.

Discussion
Currently, there are studies that point out the potential of AGR3 in breast carcinogenesis. 5,10,11,13The present systematic review and meta-analysis evaluated 713 cases and found a high prevalence of AGR3 protein expression in patients with breast cancer.The AGR3 protein was associated with positivity of estrogen and progesterone receptors, histological grade II, low expression of Ki-67, recurrence or distant metastasis, and luminal subtypes.In addition, AGR3 has a prognostic value for conferring worse OS and DFS in patients with histological grades I and II.
In the present study, we observed a prevalence of AGR3 positivity of 62% in breast cancer, which demonstrates the relevance of this protein in breast carcinogenesis, confirming the predominant expression previously reported. 5,11The expression of AGR3 in general seems to be associated with a less aggressive phenotype, with hormone receptor positivity, low histological grade, and low proliferation rate.The association of AGR3 with the positivity of estrogen and progesterone receptors demonstrates that there is a close relationship between AGR3 and the luminal subtypes. 5,11,13This interaction has often been reported 10,11,13,14 and, in conjunction with the other histological and proliferative characteristics, it suggests that AGR3 is associated with less aggressive cancers that are generally responsive to treatment and therefore have a favorable result. 5he findings of the present study indicate that the expression of AGR3 was reduced for the triple-negative subtype of breast cancer.This finding is in line with that of another recent study that analyzed AGR3 mRNA gene expression in breast cancer cell tissues. 12lthough AGR3 expression has been associated with less aggressive clinicopathological features in breast cancer, paradoxically, in the present study, we have identified the association of AGR3 expression with distant recurrence and/or metastasis and an unfavorable outcome in relation to survival, demonstrating the complexity of that molecule.In luminal subtype B, high AGR3 expression was associated with high risk of recurrence and metastasis and poor prognosis in patients with invasive breast carcinoma. 13In addition, AGR3 appears to have protumor functions in breast cancer, by regulating the adhesion and migration processes of tumor cells through the activation of Src kinases. 22he result of the meta-analysis revealed that AGR3 expression was associated with worse OS 11,13 and DFS 13 in low-and intermediate-grade cancers.Garczyk et al. 11 suggested a prooncogenic impact of AGR3 in tumors of low and intermediate histological grade, and they also highlighted the potential of AGR3 for the early detection of breast cancer, with high specificity (of 92.5%) and sensitivity (of 35%).
Regarding the therapeutic potential, a recent study 13 concluded that, in patients with luminal subtype B and histological grades I and II, the AGR3 expression conferred an unfavorable prognosis and suggested that this patients should be treated with 5-fluoropyrimidine chemotherapy, but not taxane.The authors 13 warned that AGR3 can promote tumor progression, through processes of proliferation, invasion, and resistance to chemotherapy.The present research confirmed the association of AGR3 with important features in the breast cancer clinic, such as hormone receptors, proliferation index, and prognosis.The AGR3 protein may be a biomarker of poor prognosis in low-and intermediate-grade tumors and luminal subtypes, representing an interesting tool for the clinical management of this population.Considering the recent development in cancer research, understanding the functions of AGR3 would be inevitable for the development of predictive tools for prognosis and new target therapies. 14In addition, AGR3 can serve as a biomarker in the early detection of breast cancer and to predict the clinical outcome. 14he present systematic review and meta-analysis has certain limitations.Firstly, we included only four studies, and the size of the sample of one of them was small.Secondly, although no evidence of publication bias was verified by the Egger test, exclusion of unpublished data and gray literature may have introduced selection bias in the analysis.
The present review also has strengths.Firstly, the research was conducted in five important databases in health sciences, and there was scientific rigor in the analysis process.Secondly, there was no restrictions on the year of publication and language.Thirdly, all references included were full-text articles published in peer-reviewed journals.In addition, the studies reported no conflicts of interest and were approved by ethics committees.Although the results of the present systematic review should be interpreted with caution, the evidence presented at the moment may serve as a guide for future research and also for the clinical practice.

Conclusion
The AGR3 protein may be a biomarker to predict prognosis in luminal subtypes.In addition, in patients with tumors of low and intermediate histological grades, AGR3 expression may indicate unfavorable prognosis in relation to OS and DFS.Although the present study indicates that AGR3 may be promising to predict prognosis in luminal subtypes, we highlight the need for more high-quality studies to confirm these findings, and these should be considered when making decisions regarding the prediction of diagnosis and prognosis in breast cancer.

Fig. 1
Fig. 1 Flowchart of the process of selection of studies.

Fig. 3
Fig.3Funnel plot for the studies included in the meta-analysis.

Fig. 4
Fig.4Forest plots of the prevalence (%) of anterior gradient 3 (AGR3) expression in women with breast cancer.

Fig. 5
Fig. 5 Forest plots of the hazard ratios (HRs) for survival based on the expression of AGR3.

Table 1
recurrence or distant metastasis Chart 2 Excluded articles and reason for exclusion Lei et al.AGR3 promotes estrogen receptor-positive breast cancer cell proliferation in an estrogen-dependent manner.Oncology Letters, v. 20, n. 2, p. 1,441-1,451, 2020.Prognostic Impact of AGR3 Protein Expression in Breast Cancer Moraes et al. 613 Characteristics of the studies included in the systematic review and meta-analysis Fig. 2 Summary of the authors' judgments about each item of the risk of bias assessment for each included study.Rev Bras Ginecol Obstet Vol. 45 No. 10/2023 © 2023.Federação Brasileira de Ginecologia e Obstetrícia.All rights reserved.Abbreviations: AGR3, anterior gradient 3; GRADE, Grading of Recommendations, Assessment, Development, and Evaluations.Note: Ã Invasive ductal carcinoma.GRADE Working Group grades of evidence: high quality -further research is very unlikely to change our confidence in the estimate of effect; moderate quality -further research is likely to have an important impact on our confidence in the estimate of effect and may change the estimate; low quality -further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate; and very low quality -we are very uncertain about the estimate.Rev Bras Ginecol Obstet Vol. 45 No. 10/2023 © 2023.Federação Brasileira de Ginecologia e Obstetrícia.All rights reserved.